The initiation and persistence of inflammation is tightly regulated by cell surface receptors on immune cells that function to both amplify the immune response and clear the offending agent. The persistence of inflammation may result in inflammatory disease inclusive of autoimmunity, chronic allergy and asthma. One common feature of these diseases is the central role of Fc receptors and immune complexes. A second common feature is the high levels of cytokines that serve to recruit other immune cells amplifying the response. Understanding how Fc receptors communicate with the nucleus to initiate cytokine responses and how the released cytokines contribute to inflammation is the major focus of our efforts. The immediate objective is to identify the molecular events that activate cytokine gene transcription and to evaluate whether these events might be suitable therapeutic targets for intervention of disease. We chose to work with mast cells because they are key participants in many inflammatory responses, they express the well-characterized high affinity IgE Fc receptor, and immune complex occupation of this receptor results in the production and secretion of a diverse array of cytokines. In the past year our objectives were: 1. To study the role of various signaling proteins in Fc receptor-induction of cell responses. 2. To determine in what signaling pathways the studied proteins participate. 3. To determine how critical these proteins are in induction of cytokine gene expression and cell function in response to stimulation of the IgE Fc receptor. We met these objectives in the following manner: We studied the function of the guanine nucleotide exchange factor Vav1, and a recently identified adaptor protein called Gab2 because all are expressed exclusively in hematopoietic cells. We evaluated the importance of these proteins in mast cell responses and cytokine production and where possible we evaluated their in vivo importance by in vivo activation of mast cells in gene-disrupted mice deficient in expression of these proteins. Our studies on Vav1 demonstrated that this protein is essential in the regulation of the intracellular calcium responses required for effective mast cell activation upon stimulation of the IgE Fc receptor. In vivo studies on gene-disrupted mice demonstrated that Vav1-deficiency partly diminished the in vivo response of mast cells. In vitro studies demonstrated that Vav1 controls the activation of phospholipase C gamma, a protein that generates signals required for the increase of intracellular calcium concentrations. Because calcium is required for the appropriate function of many signaling proteins, including those that activate gene expression, it was not surprising that a defect in selected cytokine genes was observed. Vav1-deficiency mainly affected the expression of IL-2 and IFN-gamma, Th1 type cytokines. Our studies on Gab2 demonstrated a novel paradigm in Fc receptor signaling. It was previously thought that all events downstream of IgE Fc receptor aggregation depended on the activity of Lyn and Syk kinases and the assembly of a LAT organized signaling complex. Our present findings demonstrate that signals can be generated that are independent of the well-characterized Lyn-Syk-LAT axis. Thus, these findings demonstrate that the IgE Fc receptor activates two independent pathways upon its aggregation. Because the proteins under study are hematopoietic cell-specific and thus may be more suitable as possible therapeutic targets we will continue to explore their function by defining the regions of these proteins critical for the observed activity. Additionally, we will identify other proteins that interact with the studied proteins that may contribute to their function, thus providing further important information for evaluation of their potential as therapeutic targets for disease intervention.